Determined DNA sequenced derived from a papillomavirus genome, their uses for in vitro diagnostic purposes and the production of antigenic compositions

ABSTRACT

The invention concerns DNA fragments derived from the genomic DNA of HPV-33. These fragments are selected from the group of fragments extending between the nucleotide extremities defined hereafter in relation to the nucleotide-numbering in FIGS. 1a and 1b respectively: 
     76-556 
     543-864 
     867-2811 
     2728-3808 
     3326-3575 
     3842-4079 
     4198-5611 
     5516-8091 
     The invention also relates to the use of these fragments as probes for the detection of HPV in tissue cultures.

This is a continuation of application Ser. No. 09/577,493, filed May 25,2000, now U.S. Pat. No. 6,242,250; which is a continuation of Ser. No.09/330,076, filed Jun. 11, 1999, issued as U.S. Pat. No. 6,107,086, Ser.No. 08/789,781, filed Jan. 28, 1997, abandoned; which is a continuationof Ser. No. 08/466,711 filed Jun. 6, 1995 issued as U.S. Pat. No.5,648,459; which is a divisional of Ser. No. 08/222,569 filed Mar. 25,1994, issued as U.S. Pat. No. 5,554,538; which is a continuation of Ser.No. 08/161,239 filed Nov. 10, 1993, abandoned; which is a continuationof Ser. No. 08/032,694 filed Mar. 17, 1993, abandoned; which is acontinuation of Ser. No. 07/908,895 filed Jul. 8, 1992, abandoned; whichis a continuation of Ser. No. 07/664,503 filed Mar. 5, 1991, abandoned;which is a continuation of Ser. No. 07/518,302 filed May 2, 1990,abandoned; which is a continuation of Ser. No. 07/128,341 filed Nov. 20,1987, abandoned; all of which are incorporated herein by reference.

The invention pertains to determined DNA sequences derived from apapillomavirus genome, more particularly DNA recombinants, includingvectors, modified by such DNA sequences in such manner that, when saidDNA recombinants are introduced in suitable host cells in which said DNArecombinants can be replicated, the said DNA sequences can be expressedin the form of the corresponding proteins. The invention further relatesto the proteins themselves, which can be purified and used for theproduction of immunogenic compositions.

The invention pertains more particularly to DNA products of thepapillomavirus designated as IP-2 (now redesignated as HPV-33) in theEuropean patent application filed under number 85.402362.9 on Nov. 29,1985, the contents of which are incorporated herein by reference. Aplasmid containing the DNA of said virus has been deposited at the CNCM(“Collection nationale de Culture de Micro-Organismes” of the PasteurInstitute of Paris) under number I-450.

Papillomaviruses are members of the papovavirus family and possess agenome of about 7,900 base pairs (bp) consisting of a covalently closedcircular DNA molecule. Human papilloma viruses (HPV) are classified onthe basis of their DNA sequence homology (6) and nearly 40 types havenow been described. Considerable insight into HPV biology and theirinvolvement in human disease has been attained by the application of thetechniques of molecular biology. A possible role for HPVs in humancancer was suspected following the detection of HPV DNA in tumorsresulting from the malignant conversion of genital warts (33). Thecloning of two HPV genomes, HPV-16 and HPV-18 (3, 11) from cervicalcarcinomas has further stimulated research in this field of immensesocio-economic importance. These viruses were discovered in more than70% of the malignant genital tumors examined and in many others HPV-16related sequences were detected (3, 16, 33). Amongst these is HPV-33which was recently cloned from an invasive cervical carcinoma usingHPV-16 as a probe under conditions of reduced stringency (1). In thepresent study we have determined the DNA sequence of HPV-33 and describeits relationship to HPV-16. Among the papillomaviruses HPV-33 is uniqueas it possesses a 78 bp tandem repeat which strongly resembles theenhancer of SV40 (4, 14).

The invention stems from the cloning strategy disclosed hereafter of thegenome of HPV-33 which enabled particular DNA sequences to beidentified, more particularly those providing hybridization probes,particularly useful for the detection of DNA of papillomaviruses relatedto HPV-33 in human tissue, whereby positive responses can be related tothe possible development in the host of invasive cervical carcinomas.

Reference is hereafter made to the drawings in which the figs concernrespectively:

FIGS. 1a and 1 b. Nucleotide sequence of HPV-33. Position 1 on thecircular genome corresponds to a “Hpa-like” sequence found by alignmentwith HPV-6b.

FIG. 2. Distribution of the major reading frames in the HPV-33 genomethe reading frames were identified by comparison with other HPVsequences and the stop codons are represdented as vertical bars. Alsoindicated are the locations of unique restriction sites (S, SmaI; E,EcoRV; B2, BglII; B1, BglI) and the likely polyadenylation signals (PA)for the early and late transcripts. In addition to these, 6 otherpotential PA sites (AATAAA) were detected at positions 662, 1215, 1221,2666, 5837 and 6239.

FIG. 3. Principle features of the non-coding region. A section of thenon-coding region from positions 7500 to 114 is shown. The 78 bp tandemrepeats are overlined and those regions resembling the Z-DNA formingelement of the SV-40 enhancer are indicated. Potential promoter elementsare denoted by stars and the 3 copies of the 12 bp palindrome enclosedbetween two rows of dots.

Preferred sequences are those which encode full proteins, moreparticularly and respectively the nucleotidic sequences having the openreading frames referred to in table I hereafter.

The conditions under which the DNA sequence analysis were performed aredefined under the heading “MATERIALS AND METHODS” hereafter. Theconclusions which were drawn from this sequence analysis appear underthe heading “DISCUSSION”.

MATERIALS AND METHODS

DNA sequence analysis, The source of HPV-33 sequenced in this study wasplasmid p15-5 (1) which consists of a BglII linearized HPV-33 genomecloned in a pBR322 derivative. A library of random DNA fragments(400-300 bp) was prepared in M13mp8 (17) after sonication and end-repairof p15-5, essentially as described previously (28). DNA sequencing wasperformed by the dideoxy chain termination method (19, 20) with themodifications of Biggin et al. (2). Most of the seQuence was derived inthis way although part of the non-coding region was found to be absentor under-represented in the M13 library (>300 clones). The sequence ofthis region was obtained directly from p15-5 using the method of Smith(24). Briefly, restriction fragments isolated from 2 “complemenary” M13clones were used to prime DNA synthesis on templates prepared from p15-5which had been linearized with a restriction enzyme and then treatedwith exonuclease III (200 units/pmol DNA for 1 h at 22° C.). Computeranalysis, DNA sequences were compiled and analysed with the programs ofStaden (26, 27) as modified by B. Caudron. Optimal alignments of DNA orprotein sequences were obtained using the algorithm developed by Wilburand Lipman (31).

RESULTS AND DISCUSSION

Genomic Arrangement of HPV-33—The complete 7909 nucleotide sequence ofHPV-33, determined by the M13 shotgun cloning/dideoxy sequencingapproach, is presented in FIG. 1. On average each position was sequenced6. 5 times. In agreement with the convention for other papillomavirussequences the numbering begins at a site resembling the recognitionsequence for HpaI in the non-coding region.

An analysis of the distribution of nonsense codons (FIG. 2) shows that,as in all other sequenced papillomaviruses, the 8 major open readingframes are located on the same strand. Some features common to HPV-33and HPV types 1 a, 6 b and 16 together with the cottontail rabbitpapillomavirus and the prototype bovine papillomavirus, BPV-1, (5, 7, 8,13, 21, 22) include the overlap between the largest open reading framesin the early region, E1 and E2, and the inclusion of E4 within thesection encoding E2. Interestingly, the BglII site used in the molecularcloning of HPV-33 is situated within the E1/E2 overlap. Another propertycommon to all papillomaviruses, except BPV-1, is the overlap between theL1 and L2 reading frames. Following L1 is the 892 bp non-coding regionwhich, by analogy with BPV1 (15, 29) undoubtedly contains the origin ofreplication and various transcriptional regulatory elements. Theprincipal characteristics of the HPV-33 genome are summarized in Table1.

Nucleotide Sequence Comparison with HPV-16—HPV-16 is the only otheroncogenic papillomavirus, isolated from tumors of the ano-genitalregion, which has been completely sequenced (22). The gross features ofHPV-33 resemble those of HPV-16 except that the El reading frame of thelatter is interrupted. All of the coding sequences in HPV-33, exceptthat of E5, are slightly shorter than their counterparts in HPV-16. Thismay contribute to the fact that its non-coding region, between L1 and E6(FIG. 2), is 76 bp longer thereby keeping the genomes nearly constant insize.

When the open reading frames were compared pairwise (Table 2) it wasfound that E1, E2, E6, E7, L1 and L2 displayed between 65-75% homologywhereas those for E4 and E5 were more divergent (about 50% homology).These findings confirm the heteroduplex analysis performed previously(1). A comparative study (8) of papillomavirus E1 gene products showedthat the polypetide consists of an NH₂-terminal segment whose sequenceis highly variable, and a COOH-terminal domain of well-conserved primarystructure. The longest stretch of perfect sequence homology, 33nucleotides (positions 1275-1307, FIG. 1) is found near the 5′-end ofthe E1 reading frame in a region encoding the variable domain of thepolypeptide. Several other regions of complete identity (19-28nucleotides) were detected elsewhere in E1, and also in E2, L2 and L1.As many of these sequences are not found in the genomes of other HPVs,such as HPV-1 a and HPV-6b, this raises the possibility that thecorresponding oligonucleotides could be produced and used as diagnostichybridization probes for screening biopsy material from potentiallytumorigenic lesions.

Potential Gene Products—The papillomavirus gene products may be dividedinto those which are believed to play a purely structural role, L1 andL2, and those required for viral propagation and persistence. Theresults of a comparison of the probable products of the major readingframes from HPVs-33, 16 and 61 b are summarized in Table 2. As expectedthere is strong identity between the ocogenic HPVs-33 and 16,particularly for the proposed E1, E6, E7, L2 and L1 proteins. Whenconservative substitutions are included the homology between the two L1polypeptides increases to 90% suggesting that the corresponding capsidsmust be antigenically related. In contrast, significantly weakerhomologies were detected when the analysis was extended to include thebenign genital wart-forming HPV-6 b (Table 2). Comparison of the HPV-16proteins with those of HPV-6 b revealed slightly more homology than wasfound with HPV-33 suggesting a closer evolutionary relationship.

The non-coding Region—The non-coding region of HPV-33 displays severalunique properties and bears only weak resemblance to its homologue inHPV-16. Located between the L1 stop codon and including the putativepolyadenylation signal for the late transcripts is a stretch of 223 bp(positions 7097-7320, FIG. 1) unusually rich in T+G (79%). Containedwithin this segment are two copies of a 19 bp direct repeat (with onemismatch) and 7 copies of the motif TTGTRTR (where R is A or G). Thelatter is also found 7 times in the corresponding region of HPV-16suggesting that it may represent a recognition site for proteinsinvolved in replication. It should be noted that nascent replicationforks have been localised in this regiion of the BPV-1 genome (29) andthat the origin of replication of the Epstein-Barr virus consists of afamily of repeated sequences (32).

A 12 bp palindrome (ACCG . . . CGGT) that occurs exclusively in thenon-coding region of all papillomavirus genomes examined was recentlyreported by Dartmann et al. (9). Three copies were found in the HPV-33genome (FIG. 3) and these occupy the same positions in the non-codingregion of HPV-16. A role for the palindrome as a possible control sitefor the early promoter was proposed (4, 9, 35 15) and indirect supportis provided by our finding that the non-coding regions of HPVs, such asHPV-33, do not display the clustered arrangement of recognition sitesfor the promoter-specific, activation factor Sp1 (12). This is in directcontrast to the situation in another papovavirus, SV40 (12, 14).

The most striking feature of HPV-33 is a perfect 78 bp tandem repeatlocated 200 bp after the putative origin of replication (FIG. 3). Noother repeats of this size or sequence have been described in thegenomes of other papillomaviruses. The presumed early promoter forHPV-33 is located about 300 bp downstream from the tandem repeat and thecharacteristic promoter elements (4) could be identified (FIG. 3). Thesize, position and arrangement of the 78 bp repeats in the HPV-33 genomesuggest that they may function as enhancers of viral transcription.Tandem repeats of 72, 73 and 68 bp have been located near the earlypromoter of SV40 (4, 14), in the LTR of moloney murine sarcoma virus(10), and in the BK virus genome (23) and shown to enhance transcriptionfrom PolII dependent promoters in a cis-active manner. From mutagenesisof the SV40 enhancer (14, 30) and sequence comparisons of characterizedtranscriptional activators a consensus enhancer sequence was derived.This structure could not be detected in the 78 bp repeat but a potentialZ-DNA forming region was uncovered. Z-DNA is believed to attractregulatory molecules to eukaryotic promoters and a Z-DNA antibodybinding site has been demonstrated within the SV40 enhancer (18). Thesequence to which this antibody binds is also found, albeit with asingle mismatch, in the putative HPV-33 enhancer (positions 7520-7527,7599-7606, FIGS. 1, 3).

The proposed HPV-33 enhancer shows no extended sequence homology to thewell-characterized enhancers nor to other papillomavirus regulatoryregions. However, it has recently been demonstrated that anenhancer-like element is located in the non-coding region of BPV-1 andthat it requires the E2 product for activation (25). These findingssupport our proposal that the 78 bp tandem repeats could have enhancerfunction and may indicate that the relatively low homology (Table 2)between the E2 proteins of HPV-33 and 16 reflects a specificity for thecorresponding enhancer/regulatory regions.

Tables 1 and 2 which have been referred to in the instant disclosurefollow.

TABLE 1 Principal features of the HPV-33 genome Open Reading FIRST STOPFrame START ATG CODON mol. wt. E6 76 109 556 TGA 17 632 E7 543 573 654TAA 10 825 E1 867 879 2811 TGA 72 387 E2 2728 2749 3808 TAA 40 207 E43326 — 3575 TAG  9 452 E5 3842 — 4079 TAA  9 385 L2 4198 4210 5611 TAG50 539 L1 5516 5594 7091 TAA 55 839

a. Calculated from the first ATG where this exists or from the start ofthe open reading frame.

TABLE 2 Comparison of HPV proteins^(a) HPVs HPVs Protein 33v16 33v6b16v6b E6 65(70) 36(51) 37 E7 61(69) 55(60) 56 E1 61(69) 50(60) 53 E253(65) 46(58) 45 E4 52(55) 39(46) 48 E5 40(52) 39(43) 33 L2 64(66)52(58) 53 L1 81(75) 68(69) 71 ^(a)-Expressed as % homology afteralignment with the program of (31). Values in parenthesis represent %nucleotide sequence homology.

The invention relates more particularly to sequences corresponding tothe open reading frames of E6, E7, E1, E2, E4, E5, L2, L1.

The invention pertains also the uses of these sequences as hybridizationprobes, either those which are useful also for the detection of otherpapillomaviruses, thus of groups of papillomaviruses—such as probescontaining part or all of the open reading frames corresponding to L1—orthose which are more virus—specific, i. e. probes containing part or allof the open reading frame corresponding to.

It also relates to other probes which detect sub-groups ofpapillomaviruses, particularly probes for the detection of viruses whichcan be related to major classes of diseases, i. e. viruses associatedwith tumors. By way of example of one of said probes one should mentionthat which contains the sequence positionned between nucleotides 1275and 1307 according to the numbering of the nucleotides in FIGS. 1A, 1B.

Needless to say that the invention also pertains to all of said DNAsequences, when labelled by a suitable label, i. e. a radioactiveenzymatic or immunofluorescent label.

DNAs derived from the viral genome and which carry nucleotides modifiedby a chemical group which can be recognized by antibodies also form partof the invention. It is well known that such DNAs can be produced bynicktranslation in the presence of nucleotides modified accordingly.These DNAs form particularly valuables hybridization probes which, whenhybridized to a DNA preparation containing the complementary strandsought, can be detected by the above mentioned antibodies.

The invention also pertains to the diagnostic methods per se. Suitablemethods are exemplified hereafter.

Several hybridization methods may be used. For example, the spothybridization method includes, after denaturation of the DNA, thedeposition of an aliquot of the DNA onto film supports (nitrocelluloseor Genescreenplus), the hybridization of each film under the usualconditions with the probe, and the detection of the radioactive hybridby contact exposition of the hybridized film onto radiographic film.Another possibility is replicated culture hybridization which involvesagarose gel electrophoresis separation of the DNA fragments resultingfrom treatment of the DNA by restriction enzymes, the transfer of thefragments after alkaline denaturation onto films (nitrocellulose orGenescreenplus) and their hybridization under usual conditions withdifferent mixtures of probes. The formation of radioactive hybrids isdetected again by contact exposition of the hybridization support filmsonto radiographic film.

For instance the probes of the invention can be used for the detectionof the relevant viruses (or DNAs thereof) in preparation consisting of abiopsy of cells obtained by scraping a lesion, or of biopsy sectionsfixed with Carnoy's mixture (ethanol, chloroform, acetic acid 6:3:1) andincluded in paraffin.

The above nucleotide sequences can be inserted in vectors, to providemodified vectors which, when introduced in the suitable cell host, arecapable of providing for the transcription and, where appropriate,translation of said DNA sequences to produce the corresponding proteinswhich can then be isolated from cellular extracts of the hosts.Obviously it is within the knowledge of the man skilled in the art toselect the appropriate vectors, particularly in relation to the host tobe transformed therewith. Vectors consist for instance of plasmids orphages which will be selected according to their recognized capabilityof replicating in the corresponding procaryotic cells (or yeast cells)and of allowing for the expression of the DNA sequence which they carry.

The invention also relates to DNA recombinants containing an insertconsisting of a DNA sequence corresponding to any of the above-definedopen reading frames or of a part thereof, and suitably engineered toallow for the expression of the insert in eucaryotic cells, particularlycells of warm-blooded animal. Suitable DNA recombinants are geneticconstructs in which said insert has been placed under the control of aviral or eucaryotic promoter recognized by the polymerases of theselected cells and which further comprise suitable polyadenylation sitesdownstream of said insert.

By way of example, the invention pertains to DNA recombinants containingany of the above-mentioned open-reading inserts placed under the controlof a promoter derived from the genome of the SV40 virus. Such DNArecombinants—or vectors—can be used for the transformation of highereucaryotic cells, particularly cells of mammals (for instance Verocells). The invention further pertains to portions of the aboveidentified DNA sequences which, when inserted in similar vectors, areable to code for portions of the corresponding proteins which haveimmunological properties similar to those encoded by the full nucleotidesequences mentioned above. The similarity of immunological propertiescan be recognized by the capacity of the corresponding polypeptidesproduced by the relevant host to be recognized by antibodies previouslyformed against the proteins produced by the cells previously transformedwith vectors containing the above mentioned entire DNA sequences.

It goes without saying that the invention also pertains to anynucleotidic sequence related to the preceding ones which may be obtainedat least in part synthetically, and in which the nucleotides may varywithin the constrainsts of the genetic code, to the extent where thesevariations do not entail a substantial modification of the polypeptidicsequences encoded by the so-modified nucleotidic sequences.

It already flows from the preceding discussion that the invention alsopertains to the purified proteins or polypeptides themselves asobtainable by the methods discussed hereabove. These polypeptides, whenproduced in a suitable host, can either be obtained from the cells, forinstance after rupturing of their cell walls, or from the culture mediumof said cells when excreted in said cell medium, depending on the cellDNA recombinant system which is used. The polypeptide obtained can thenbe purified by resorting to usual purification procedures. It should beunderstood that “purified” in the instant context means a level ofpurity such that, when electrophoresed in SDS-PAGE, the purifiedproteins yield a single detectable band, say by western blot.

The viral proteins obtained, more particularly the structural proteins,for instance as a result of the expression of said DNA sequences in E.coli, can be used for the in vitro detection of antibodies againstpapillomavirus likely to be detected in tissue samples of patientspossibly infected with papillomavirus.

Of particular relevance are the genetically engineered proteins havingthe peptidic sequences which can be deduced from the L1 and L2 openreading frames. Another peptide of interest is the E6 ^(*) protein (E6star), the synthesis of which can be induced by splicing and whichencoded by a nucleotidic sequence located between nucleotides 229 (donorsite) and 404 (acceptor site) of the HPV 33 sequence (see moreparticularly FIG. 1A), which sites also define the putative splicingsites in the E6 ^(*) open reading frame of HPV 33. Reference may be hadto the publication of Schneider-Gardicke and Schwartz, Embo. J. , 5,2285-2292, as concerns the conditions of the production of suchproteins.

These purified polypeptides can in turn be used for the production ofcorresponding antibodies which can be used for diagnosing in vitro thepresence of viral polypeptides in a biological fluid, particularly in aserum or tissue culture of a patient. Like in the preceding instance,the invention relates to portions of the above defined polypeptides,particularly those which are recognized by the same antibodies or to thecontrary are able to elicit in vivo the production of antibodiesrecognizing the complete proteins.

It must be understood that the inventions relates also specifically tothe particular peptides encoded by the DNA regions specifically referredto in the preceding disclosure and which have been found of particularinterest.

The invention further concerns host cells transformed with DNArecombinants containing nucleotidic sequences directing the expressionof the different peptides mentioned hereabove, and effectively capableto produce said peptides when cultured in an appropriate culture medium.

The invention finally also pertains more particularly to the antibodiesthemselves which can be obtained from an animal, such as rabbit,immunized in standard manner with said purified polypeptides and/or fromhybridomas previously prepared also in any known manner. Of particularinerest are the antibodies (polyclonal and monoclonal antibodies)directed against the strutural proteins. These antibodies are useful forthe detection of viral infection. The antibodies which recognize the L1,L2 and E6 ^(*) proteins of HPV-33 are of particular significance.Antibodies specific of L2 provide diagnostic tools for the in vitrodetection of specific viruses sharing with HPV-33 a sequence encoding asimilar L2 protein. Antibodies specific to L1 are useful for thedetection of the groups of viruses, to which HPV-33 belongs. Antibodiesspecific to the E6 ^(*) protein are useful for the detection of theoncogenic character of the virus causing the abovesaid viral infection.

The invention also relates to intergenic sequences of particularinterest, particular the 78 bp sequence. This sequence is of particularinterest as a possible insert in eucaryotic vectors, particularly in aposition upstream of the promoter and downstream of the site at whichtranscription of the gene or nucleotide sequence the transcription ofwhich is sought is initiated in the relevant host.

All documents referred to herein are incorporated herein by reference.Particularly these documents can be referred to as concerns thedefinition of expressions used in this application where appropriate. Assuch they form part of the present disclosure.

BIBLIOGRAPHY

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What is claimed is:
 1. A process for producing an immune response to apapillomavirus antigen in a mammal comprising: administering to themammal an HPV-33 papillomavirus peptide selected from the groupconsisting of E1, E2, E4, E5, E6, E7, L1, L2, and fragments of any ofthe foregoing peptides, wherein administration results in an immuneresponse.
 2. The process of claim 1, comprising administering E1 or afragment thereof.
 3. The process of claim 1, comprising administering E2or a fragment thereof.
 4. The process of claim 1, comprisingadministering E4 or a fragment thereof.
 5. The process of claim 1,comprising administering E5 or a fragment thereof.
 6. The process ofclaim 1, comprising administering E6 or a fragment thereof.
 7. Theprocess of claim 1, comprising administering E7 or a fragment thereof.8. The process of claim 1, comprising administering L1 or a fragmentthereof.
 9. The process of claim 1, comprising administering L2 or afragment thereof.
 10. The process of claim 2, comprising administering apeptide having a molecular weight of about 72.4 kilodaltons or less. 11.The process of claim 3, comprising administering a peptide having amolecular weight of about 40.2 kilodaltons or less.
 12. The process ofclaim 4, comprising administering a peptide having a molecular weight ofabout 9.5 kilodaltons or less.
 13. The process of claim 5, comprisingadministering a peptide having a molecular weight of about 9.4kilodaltons or less.
 14. The process of claim 6, comprisingadministering a peptide having a molecular weight of about 17.6kilodaltons or less.
 15. The process of claim 7, comprisingadministering a peptide having a molecular weight of about 10.8kilodaltons or less.
 16. The process of claim 8, comprisingadministering a peptide having a molecular weight of about 55.8kilodaltons or less.
 17. The process of claim 9, comprisingadministering a peptide having a molecular weight of about 50.5kilodaltons or less.
 18. The process of claim 1, wherein the peptide isproduced using recombinant DNA technology.
 19. The process of claim 18,wherein the peptide is produced in E. coli.
 20. A process for obtainingantibodies from a mammal comprising: administering to a mammal an HPV-33papillomavirus peptide selected from the group consisting of E1, E2, E4,E5, E6, E7, L1, L2, and fragments of any of the foregoing peptides,wherein administration results in an immune response, and obtainingantibodies from the mammal in which the immune response has occurred.21. The process of claim 20, wherein obtaining antibodies comprises:collecting whole blood or serum from the mammal in which the immuneresponse has occurred, separating a soluble fraction from an insolublefraction of the whole blood or serum, and isolating antibodies from theinsoluble or soluble fraction.
 22. The process of claim 21, whereinisolating antibodies involves affinity purification.
 23. The process ofclaim 22, wherein affinity purification is performed with E1or afragment thereof.
 24. The process of claim 22, wherein affinitypurification is performed with E2 or a fragment thereof.
 25. The processof claim 22, wherein affinity purification is performed with E4 or afragment thereof.
 26. The process of claim 22, wherein affinitypurification is performed with E5 or a fragment thereof.
 27. The processof claim 22, wherein affinity purification is performed with E6 or afragment thereof.
 28. The process of claim 22, wherein affinitypurification is performed with E7 or a fragment thereof.
 29. The processof claim 22, wherein affinity purification is performed with L1 or afragment thereof.
 30. The process of claim 22, wherein affinitypurification is performed with L2 or a fragment thereof.